This invention is directed to methods of regioselective alkylation of pentosyl sugar moieties at the 2xe2x80x2-OH position. The present invention is further directed to reduction and derivatization of the 2xe2x80x2-O-alkylated compounds produced by these methods. The methods are useful for the preparation of 2xe2x80x2-O-alkyl nucleotides, nucleosides and nucleoside surrogates that are useful as precursors for the preparation of oligomeric compounds. Such oligomeric compounds are useful as therapeutics, diagnostics, and research reagents.
In certain embodiments of the invention, the inclusion of one or more 2xe2x80x2-O-aminooxyethyl moieties in an oligonucleotide provides, inter alia, improved binding of the oligonucleotide to a complementary strand. In further embodiments of the invention, the inclusion of one or more 2xe2x80x2-O-aminooxyethyl moieties in an oligomeric compound of the invention provides one or more conjugation sites useful for the conjugation of various useful ligands. Such ligands include, for example, reporter groups and groups for include, for example, reporter groups and groups for modifying uptake, distribution or other pharmacodynamic properties. The specific objectives and advantages of the present invention will become apparent to the art-skilled from the description of the preferred embodiments of the invention.
It has been recognized that oligonucleotides and oligonucleotide analogs (oligomeric compounds) can be used to modulate mRNA expression by a mechanism that involves the complementary hybridization of relatively short oligonucleotides to mRNA such that the normal and essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence-specific base pair hydrogen bonding of an oligonucleotide to a complementary RNA or DNA.
Oligonucleotides are used as diagnostics, therapeutics and as research reagents. For this, the ability of an oligonucleotide to bind to a specific DNA or RNA with fidelity is an important factor. The relative ability of an oligonucleotide to bind to complementary nucleic acids is compared by determining the melting temperature of a particular hybridization complex. The melting temperature (Tm), a characteristic physical property of double helices, is the temperature (in xc2x0 C.) at which 50% helical versus coil (unhybridized) forms are present. Tm is measured by using UV spectroscopy to determine the formation and breakdown (melting) of hybridization. Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently, a reduction in UV absorption indicates a higher Tm. The higher the Tm, the greater the strength of the binding of the nucleic acid strands. Therefore, oligonucleotides modified to hybridize with appropriate strength and fidelity to targeted RNA (or DNA) are greatly desired for use as research reagents, diagnostic agents, and as oligonucleotide therapeutics.
Various modifications to the base, sugar and internucleoside linkage have been introduced into oligonucleotides at selected positions, and the resultant effect relative to the unmodified oligonucleotide compared. A number of modifications have been shown to improve one or more aspects of the oligonucleotide. Useful 2xe2x80x2-modifications that have been shown to improve aspects of oligonucleotides include halo, alkoxy and allyloxy groups. Many 2xe2x80x2-O-modified oligonucleotides having increased hybridization and nuclease resistance have been used in antisense research.
The use of antisense compounds as drug candidates with potential clinical applications requires that they form stable duplexes with target mRNA""s, prevent translation of messages (most often via RNase H-mediated cleavage), and have resistance to nucleases. Phosphorothioate backbone modified oligonucleotides having 2xe2x80x2-O-modified monomers at selected positions have been reported to be effective antisense molecules. Cook, P. D., 1998, Second Generation 2xe2x80x2-Modified Antisense Oligonucleotides, J. A. Bristol (Ed.), Annu. Rep. Med. Chem., Vol. 33, pp. 313-325, Academic Press, New York. The phosphorothioate internucleoside linkage enhances nuclease resistance, while the 2xe2x80x2-O-modification increases hybridization. Superior antisense activity has been shown for 2xe2x80x2-O-modified oligomeric compounds. Martin, Helv. Chim. Acta., 1995, 78, 486-504. These oligonucleotides were prepared using xe2x80x9cgapmerxe2x80x9d technology. Monia et al., J. Biol. Chem., 1993, 268, 14514-14522. Gapmer technology utilizes nuclease-resistant internucleoside linkages at selected positions while using native or other internucleoside linkages at internal positions. Generally, the 3xe2x80x2 and 5xe2x80x2 regions of the oligomeric compound will have contiguous internucleoside linkages providing superior nuclease resistance while the internal region may have native or other internucleoside linkages.
In addition to 2xe2x80x2-O-methoxyethyl modified oligomeric compounds, oligomeric compounds having pseudoisosteres of 2xe2x80x2-O-methoxyethyl modification have also shown superior hybridization qualities. Included in this group of 2xe2x80x2-O-modifications is the 2xe2x80x2-O-aminooxyethyl (AOE) modification. Kawasaki et al., 1998, Synthesis, Hybridization, and Nuclease Resistance Properties of 2xe2x80x2-O-Aminooxyethyl Modified Oligonucleotides, G. Gosselin and B. Rayner (Eds.), XIII International Round Table, Nucleosides, Nucleotides, and their Biological Applications, Montpellier, France. The hydroxylamino function present in this modification is observed in nature in the form of glycosylated antibiotics. Walker et al., J. Am. Chem., 1994, 116, 3197-3206. The hydroxylamino function has also been synthetically incorporated into oligonucleotide backbones. Peoc""h et al., Nucleosides Nucleotides, 1997, 16, 959-962. Among the unique properties of the hydroxylamino function are the unusual conformational preferences of the Nxe2x80x94O bond and the surprisingly low pKa (MeONH2, 4.2, MeONHMe, 4.75, MeONHMe2, 3.65).
Ikehara et al. (European J. Biochem., 1984, 139, 447) have reported the synthesis of a mixed octamer containing one 2xe2x80x2-deoxy-2xe2x80x2-fluoroguanosine residue or one 2xe2x80x2-deoxy-2xe2x80x2-fluoroadenine residue. Guschlbauer and Jankowski (Nucleic Acids Res, 1980, 8, 1421) have shown that the contribution of the C3xe2x80x2-endo conformer increases with ISIS-5040 -5-increasing electronegativity of the 2xe2x80x2-substituent. Thus, 2xe2x80x2-deoxy-2xe2x80x2-fluorouridine contains 85% of the C3xe2x80x2-endo conformer.
Furthermore, evidence has been presented which indicates that 2xe2x80x2-substituted-2xe2x80x2-deoxyadenosine polynucleotides resemble double-stranded RNA rather than DNA. Ikehara et al. (Nucleic Acids Res., 1978, 5, 3315) have shown that a 2xe2x80x2-fluoro substituent in poly A, poly I, or poly C duplexed to its complement is significantly more stable than the ribonucleotide or deoxyribonucleotide poly duplex as determined by standard melting assays. Ikehara et al. (Nucleic Acids Res., 1978, 4, 4249) have shown that a 2xe2x80x2-chloro or -bromo substituent in poly(2xe2x80x2-deoxyadenylic acid) provides nuclease resistance. Eckstein et al. (Biochemistry, 1972, 11, 4336) have reported that poly-(2xe2x80x2-chloro-2xe2x80x2-deoxy-uridylic acid) and poly(2xe2x80x2-chloro-2xe2x80x2-deoxycytidylic acid) are resistant to various nucleases. Inoue et al. (Nucleic Acids Res., 1987, 15, 6131) have described the synthesis of mixed oligonucleotide sequences containing 2xe2x80x2-OMe substituents on every nucleotide. The mixed 2xe2x80x2-OMe-substituted oligonucleotide hybridized to its RNA complement as strongly as the RNA-RNA duplex which is significantly stronger than the same sequence RNA-DNA hetero duplex (Tris, 49.0 and 50.1 versus 33.0 degrees for nonamers) Shibahara et al. (Nucleic Acids Res., 1987, 17, 239) have reported the synthesis of mixed oligonucleotides containing 2xe2x80x2-OMe substituents on every nucleotide. The mixed 2xe2x80x2-OMe-substituted oligonucleotides were designed to inhibit HIV replication.
It is believed that the composite of the hydroxyl group""s steric effect, its hydrogen bonding capabilities, and its electronegativity versus the properties of the hydrogen atom is responsible for the gross structural difference between RNA and DNA. Thermal melting studies indicate that the order of duplex stability (hybridization) of 2xe2x80x2-methoxy oligonucleotides is in the order of RNA-RNA greater than RNA-DNA greater than DNA-DNA.
International Publication Number WO 91/06556, published May 16, 1991, and U.S. Pat. No. 5,466,786 disclose oligomers derivatized at the 2xe2x80x2-position with substituents. These oligomers are stable to nuclease activity. Specific 2xe2x80x2-O-substituents which were incorporated into oligonucleotides include ethoxycarbonylmethyl (ester form), and its acid, amide and substituted amide forms.
Martin (Helvetica Chimica Acta, 78, 1995, 486-504) discloses certain nucleosides, and oligonucleotides prepared therefrom, that include 2xe2x80x2-methoxyethoxy, 2xe2x80x2-methoxy(tris-ethoxy) and other substituents. Oligonucleotides containing nucleosides substituted with either the 2xe2x80x2-methoxyethoxy and 2xe2x80x2-methoxy(tris-ethoxy)substituents exhibited improved hybridization, as judged by increase in Tm.
The use of esters, such as methyl bromoacetate, as electrophiles for the alkylation of nucleosides, have been used where the sugar moiety of the nucleosides being alkylated has been selectively protected. See, PCT application WO 91/06556, entitled xe2x80x9c21-modified oligonucleotides,xe2x80x9d filed Oct. 24, 1990; and Keller et al., Helv. Chim. Acta., 1993, 76, 884-892.
It has been recognized that oligomeric compounds having improved hybridization and nuclease resistance are of great importance in the development of useful research reagents, diagnostic agents and therapeutic agents. There exists a need in the art for improved processes, for the preparation of 2xe2x80x2-O-modified nucleosidic oligomeric compounds, which are more facile, are faster and are cheaper than processes currently known in the art.
The present invention provides methods for the regioselective alkylation at the 2xe2x80x2-hydroxy position of a sugar moiety of a nucleoside. These methods are useful for the synthesis of 2xe2x80x2-O-alkyl nucleotides, which in turn serve as precursors in the preparation of oligomeric compounds. The methods of the present invention use nucleosides bearing unprotected 2xe2x80x2- and 3xe2x80x2-hydroxyl functionalities. These nucleosides are treated with a base and alkylated with the reactive form of an ester to form 2xe2x80x2-O-modified nucleosides.
The present invention provides methods for the preparation of a compound of formula: 
wherein:
Q is H or a hydroxyl protecting group;
Bx is a heterocyclic base moiety; and
Z is C1 to C12 alkyl;
comprising the steps of:
(a) selecting a compound of formula: 
(b) dissolving said compound in at least one solvent to form a solution;
(c) cooling said solution to a temperature of from about 5xc2x0 C. to about minus 50xc2x0 C.;
(d) treating said cooled solution with a base to give a mixture;
(e) warming said mixture to a temperature of from about minus 30xc2x0 C. to 35xc2x0 C.;
(f) cooling said mixture of step (e) to a temperature of from about 5xc2x0 C. to about minus 50xc2x0 C.; and
(g) reacting said cooled mixture of step (f) with an ester of the formula: 
where in:
Z is as defined above; and
J is a leaving group;
to give said compound.
In a preferred embodiment the heterocyclic base moiety is N3-protected-5-methyluridine, N3-protected-uridine, cytidine, 5-methylcytidine, guanosine, adenosine, or 2,6-diaminopurineriboside.
In another preferred embodiment the base is 1,5-diazabicyclo[4.3.0]non-5-ene, 1,4-diazabicyclo[2.2.2]octane or 1,8-diazabicyclo[5.4.0]undec-7-ene. In yet another preferred embodiment the base is a metal hydride, a metal hydroxide or a metal carbonate. In one preferred embodiment, the metal hydride is sodium hydride, lithium hydride or potassium hydride. In another preferred embodiment, the metal hydroxide is sodium hydroxide, potassium hydroxide or lithium hydroxide. In yet another preferred embodiment, the metal carbonate is sodium carbonate, potassium carbonate or cesium carbonate.
In a further embodiment of the present invention, the solvent is an aprotic solvent. It is preferred that the solvent be dimethylformamide, dimethylsulfoxide, dimethylacetamide, acetonitrile or hexamethylphosphoramide. It is further preferred that a combination of these solvents be used in the methods of the present invention. Thus it is preferred that the solvent used in the present invention be at least two of dimethylformamide, dimethylsulfoxide, dimethylacetamide, acetonitrile and hexamethylphosphoramide. Most preferably, the solvent is dimethylformamide having from 1% to about 40% dimethylsulfoxide.
It is preferred that the ester be an alkyl haloalkylate. It is further preferred that the ester be an alkyl bromoalkylate. It is most preferred that the ester be methyl bromoacetate.
It is preferred that the cooling of the mixture of the nucleoside and base be to a temperature of from about 5xc2x0 C. to about minus 50xc2x0 C. It is further preferred that the cooling of said mixture be to a temperature of from about minus 30xc2x0 C. to about minus 50xc2x0 C. It is still further preferred that the cooling of said mixture be to a temperature of from about minus 40xc2x0 C. to about minus 50xc2x0 C.
The present invention further provides methods for the preparation of a further compound of formula: 
wherein:
Q is H or a hydroxyl protecting group; and
Bx is a heterocyclic base moiety;
comprising treating a compound of formula: 
wherein:
Q is H or a hydroxyl protecting group;
Bx is a heterocyclic base moiety; and
Z is C1 to C12 alkyl;
with a reducing agent under suitable conditions of time, temperature and pressure.
In a preferred embodiment, the reducing agent is sodium borohydride, lithium borohydride or borane.
The present invention also provides methods for the preparation of a derivative compound of formula: 
wherein:
Q is H or a hydroxyl protecting group;
Bx is a heterocyclic base moiety;
L is C1 to C10 alkyl, xe2x80x94N(R1)R2, or xe2x80x94Nxe2x95x90C(R1)(R2);
each R1 and R2 is, independently, H, C1-C10, alkyl, a
nitrogen protecting group, or R1 and R2, together, are a nitrogen protecting group, or R1 and R2, together, are joined in a ring structure wherein said ring structure comprises at least one heteroatom selected from N and O.
In a preferred embodiment L is xe2x80x94CH3 or xe2x80x94N(CH3)CH3. In another preferred embodiment xe2x80x94N(R1)R2 is phthalimido or piperidinyl. In yet another preferred embodiment R1 and R2 are joined in a ring structure wherein said ring structure comprises at least one heteroatom selected from N and O.
The present invention provides methods of preferential regioselective alkylation of nucleosides at 2xe2x80x2-hydroxyl positions over 3xe2x80x2-hydroxyl positions. The present methods eliminate protection and subsequent deprotection steps when alkylating the 2xe2x80x2-O-position of nucleosides. The resultant 2xe2x80x2-O-alkylated nucleoside is further reduced to the intermediate 2xe2x80x2-O-hydroxyethyl nucleoside which is further derivatized to 2xe2x80x2-O-modified nucleosides of formula: 
wherein:
Q is H or a hydroxyl protecting group;
Bx is a heterocyclic base moiety; and
L is C1 to C10 alkyl, xe2x80x94N(R1)R2, or xe2x80x94Nxe2x95x90C(R1)(R2)
The present invention provides improved methods of preparing nucleosides that are useful in the preparation of oligomeric compounds possessing superior hybridization properties. Structure-activity relationship studies have revealed that an increase in binding (Tm) of certain 2xe2x80x2-sugar modified oligonucleotides to an RNA target (complement) correlates with an increased xe2x80x9cAxe2x80x9d type conformation of the heteroduplex. Furthermore, absolute fidelity of the modified oligonucleotides is maintained. Increased binding of 2xe2x80x2-sugar modified sequence-specific oligonucleotides of the invention provides superior potency and specificity compared to phosphorus-modified oligonucleotides such as methyl phosphonates, phosphate triesters and phosphoramidates as known in the literature.
Oligomeric compounds incorporating 2xe2x80x2-O-modified nucleosides of the invention are synthesized by standard solid phase nucleic acid synthesis using automated synthesizers such as Model 380B (Perkin-Elmer/Applied Biosystems) or MilliGen/Biosearch 7500 or 8800. Triester, phosphoramidite, or hydrogen phosphonate coupling chemistries (Oligonucleotides: Antisense Inhibitors of Gene Expression. M. Caruthers, p. 7, J. S. Cohen (Ed.), CRC Press, Boca Raton, Fla., 1989) are used with these synthesizers to provide the desired oligonucleotides. The Beaucage reagent (J. Amer. Chem. Soc., 1990, 112, 1253) or elemental sulfur (Beaucage et al., Tet. Lett., 1981, 22, 1859) is used with phosphoramidite or hydrogen phosphonate chemistries to provide 2xe2x80x2-substituted phosphorothioate oligonucleotides.
Unprotected nucleosides are regioselectively alkylated using methods of the present invention. Direct alkylation is faster and less expensive eliminating unnecessary protection, deprotection and purification steps required with other previously reported methods. There has been ample work published on this subject involving activated (Takaku et al., Chem. Lett., 1982, 189-192) and unactivated (Manoharan et al., Tetrahedron Lett., 1991, 32, 7171-7174) electrophiles. The degree of selectivity encompasses a wide range and appears to depend on the substrate and/or the electrophile.
The regioselectivity of the 2xe2x80x2- over the 3xe2x80x2-position of a nucleoside having unprotected 2xe2x80x2- and 3xe2x80x2-hydroxyl groups was initially determined using adenosine and 2,6-diaminopurin-9-yl-riboside. When the unprotected adenosine or 2,6-diaminopurin-9-yl-riboside was reacted with methyl 2-bromoacetate under basic conditions, at low temperatures (approximately xe2x88x9240xc2x0 C.), a highly regioselective alkylation of the 2xe2x80x2- versus 3xe2x80x2-hydroxyl position occurred. The regioselectivity for the 2xe2x80x2- over the 3xe2x80x2-position was in a ratio of about 9:1 or better. The resulting products were 2xe2x80x2-O-(methoxycarbonylmethylene)-adenosine and 2xe2x80x2-O-(methoxycarbonylmethylene)-2,6-diaminopurin-9-yl-riboside in about 75% yields. Alkylation of the purine ring was not observed in either of the syntheses, and the regioselectivity in both cases was confirmed by 2D NMR (TOCSY). This highly regioselective alkylation of the unprotected ribosides is thought to be influenced by the effects of the carbonyl group adjacent to the reactive site in this SN2 reaction. Carey, F. A., and Sundberg, R. J., Advanced Organic Chemistry, Part A: Structures and Mechanisms, 1990, 3rd ed., pp. 296-297, Plenum Press, New York, N.Y. The regioselective alkylation reactions were reproduced on multigram scales (25 g), and the small amounts of 3xe2x80x2-O-isomer were readily resolved by chromatography after alkylation or at a subsequent step.
In the context of this invention, the term xe2x80x9coligomeric compoundxe2x80x9d refers to a plurality of nucleoside monomers joined together in a specific sequence. The nucleosides of use in the present invention may be naturally-occurring or non-naturally occurring. Preferred nucleosides each have a nucleobase attached to a pentose sugar moiety and form oligomeric compounds via phosphorus linkages connecting the sugar moieties. Representative heterocyclic base moities, or nucleobases, include adenine, guanine, adenine, cytosine, uracil, thymine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, other aza and deaza thymidines, other aza and deaza cytosines, other aza and deaza adenines, other aza and deaza guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.
2xe2x80x2-O-Modified nucleosides prepared according to the methods of the present invention are further treated with reagents, using methods well known in the art and illustrated in the examples below, to convert the nucleosides into nucleoside surrogates. Nucleoside surrogates, such as DMT phosphoramidites shown in the examples below, are ready for use in standard oligonucleotide synthesis following well- established protocols. Nucleoside surrogates can include appropriate activated phosphorous atoms in PIII or PV valence states for incorporation into an oligomeric compound. Such activated phosphorous atoms include phosphoramidites, hydrogen phosphonates and triesters. The nucleoside surrogates can also include appropriate hydroxyl blocking groups including, but not limited to, dimethoxytrityl, trimethoxytrityl, monomethoxytrityl and trityl blocking groups, and other blocking groups as are known in the art.
In positioning one of the nucleoside surrogate groups of the invention in an oligonucleotide, an appropriate blocked and activated nucleoside surrogate is incorporated in the oligonucleotides in the standard manner for incorporation of a normal blocked and active standard nucleotide. As for instance, an 2xe2x80x2-O-nucleoside surrogate is selected that has an aminooxy moiety which is protected utilizing a phthalimido protecting group. One of the hydroxyl groups of the surrogate molecule is protected utilizing a dimethoxytrityl protecting group (a DMT protecting group) and the other hydroxyl group is present as a cyanoethoxy diisopropyl phosphoramidite moiety. The surrogate unit is added to the growing oligonucleotide by treating with the normal activating agents, as is known in the art, to react the phosphoramidite moiety with the growing oligomeric compound. This is followed by removal of the DMT group in the standard manner, as is known in the art, and continuation of elongation.
There are a number of modifications that can be made to nucleosides in combination with the 2xe2x80x2-O-modifications of the invention. Representative modifications that can be made to the sugar, base, or to the phosphate group of nucleosides are disclosed in International Publication Numbers WO 91/10671, published Jul. 25, 1991, WO 92/02258, published Feb. 20, 1992, WO 92/03568, published Mar. 5, 1992, and U.S. Pat. Nos. 5,138,045, 5,218,105, 5,223,618 5,359,044, 5,378,825, 5,386,023, 5,457,191, 5,459,255, 5,489,677, 5,506,351, 5,541,307, 5,543,507, 5,571,902, 5,578,718, 5,587,361, and 5,587,469, all assigned to the assignee of this application. The disclosures of each of the above referenced publications are herein incorporated by reference.
The attachment of conjugate groups to oligonucleotides and analogs thereof is well documented in the art. Compounds of the present invention include compounds bearing conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include inter-calators, reporter molecules, polyamines, polyamides, poly-ethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, phospho-lipids, biotin, phenazine, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of the present invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US 92/09196, filed Oct. 23, 1992, U.S. Pat. No. 5,578,718, and U.S. Pat. No. 5,218,105. Each of the foregoing is commonly assigned with this application. The entire disclosure of each is incorporated herein by reference.
Cleavage of oligonucleotides by nucleolytic enzymes requires the formation of an enzyme-substrate complex or, in particular, a nuclease-oligonucleotide complex. The nuclease enzymes will generally require specific binding sites located on the oligonucleotides for appropriate attachment. If the oligonucleotide binding sites are removed or blocked, such that nucleases are unable to attach to the oligonucleotides, the oligonucleotides will be nuclease resistant. In the case of restriction endonucleases that cleave sequence-specific palindromic double-stranded DNA, certain binding sites such as the ring nitrogen in the 3- and 7-positions have been identified as required binding sites. Removal of one or more of these sites or sterically blocking approach of the nuclease to these particular positions within the oligonucleotide has provided various levels of resistance to specific nucleases.
Sugars having O-substitutions on the ribosyl ring are also amenable to the present invention. Representative substitutions for a ring oxygen include S, CH2, CHF, and CF2. See, e.g., Secrist et al., Abstract 21, Program and Abstracts, Tenth International Roundtable, Nucleosides, Nucleotides and their Biological Applications, Park City, Utah, Sep. 16-20, 1992, hereby incorporated by reference in its entirety.
Strong bases amenable to the present methods include NaH, 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 1,4-diazabicyclo[2.2.2]octane (Dabco), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and CsCO3. Other aprotic polar organic solvents may also be used in place of dimethylformamide (DMF), e.g., dimethylsulfoxide, dimethyl-acetamide, acetonitrile, or hexamethylphosphoramide (HMPA).
The present invention provides methods for regioselective alkylation of a nucleoside of the formula: 
wherein Q and Bx are as described previously.
Selected nucleosides were initially dissolved in one or more solvents. One solvent that worked well was DMF. With insoluble nucleosides, dissolution is first accomplished with gentle heating in DMSO followed by dilution with DMF. In Example 11 below, 2xe2x80x2-O-(methoxycarbonylmethylene)-2,6-diaminopurin-9-yl-riboside was first dissolved in DMSO, with warming, followed by addition of DMF to give 20% DMSO in DMF as the final composition. There are many other solvents and solvent systems that are amenable to the present invention. Some representative solvents include dimethylformamide, dimethylsulfoxide, dimethylacetamide, acetonitrile, or hexamethylphosphoramide and combinations of dimethylformamide, dimethylsulfoxide, dimethylacetamide, acetonitrile, or hexamethylphosphoramide. The solvent used in the methods of the present invention may also be a combination of two or more solvents selected from those stated above.
The solution having the dissolved nucleoside is then cooled to from about 5xc2x0 C. to about minus 50xc2x0 C. and treated with a base. Sodium hydride proved effective for a number of reactions that were performed. Other representative bases that are amenable to the methods of the present invention include 1,5-diazabicyclo[4.3.0]non-5-ene, 1,4-diazabicyclo[2.2.2]-octane, 1,8-diazabicyclo[5.4.0]undec-7-ene, or cesium carbonate. The mixture resulting after addition of the base is allowed to warm to from about minus 30xc2x0 C. to about 35xc2x0 C., and then recooled to from about minus 30xc2x0 C. to about minus 50xc2x0 C. A preferred range is from about minus 40xc2x0 C. to about minus 50xc2x0 C.
The cooled mixture is next treated with an ester of formula: 
wherein:
Z is C1 to C12 alkyl; and
J is a leaving group.
An ester bearing a leaving group at its xcex1-CH2 position is the reactive form of the ester. This leaving group is cleaved off the reactive ester when the 2xe2x80x2-hydroxy group of the sugar moiety attacks the xcex1-CH2 group of the ester. This results in the formation of a covalent bond between the oxygen atom of the hydroxy group of the sugar moiety and the xcex1-CH2 group of the ester.
Leaving groups are routinely used in the art, and include halo, alkoxy, tosylate, brosylate, nosylate, mesylate and triflate. A preferred leaving group is halo. It is more preferred that the leaving group be bromo. A number of esters have been used successfully. It is preferred that the ester used in the methods of the present invention be an alkyl haloalkylate. It is further preferred that the ester be an alkyl bromoalkylate. It is most preferred that the ester be methyl bromoacetate. After the addition of ester, the reaction mixture is allowed to gradually warm to ambient temperature. After stirring at ambient temperature, the reaction mixture is worked up to give the desired 2xe2x80x2-O-esterified nucleoside of formula: 
wherein Q, Bx, and L are as previously described.
The 2xe2x80x2-O-esterified nucleoside is further reduced to give the 2xe2x80x2-O-hydroxyethyl compound. A preferred reducing agent is sodium borohydride. A number of other reducing agents are amenable to the present invention, including lithium borohydride and borane. The resultant 2xe2x80x2-O-hydroxyethyl nucleosides are further reacted with reagents to form derivative compounds such as 2xe2x80x2-O-methoxyethyl (2xe2x80x2-O-MOE) and 2xe2x80x2-O-dimethylaminoethyl (DMAOE) nucleosides. The preparation of derivative nucleosides from the 2xe2x80x2-O-hydroxyethyl nucleoside is illustrated in the examples below, and are further disclosed in PCT application PCT/US 98/02405, entitled xe2x80x9cAminooxy-Modified Oligonucleotides,xe2x80x9d filed Feb. 13, 1998.
Nucleosides prepared by the methods of the present invention are useful in the preparation of oligomeric compounds which are utilized as diagnostics, therapeutics and as research reagents and kits. The oligomeric compounds can be utilized in pharmaceutical compositions by adding an effective amount to a suitable pharmaceutically acceptable diluent or carrier. The oligomeric compounds prepared according to the methods of the present invention can be used for treating organisms having a disease characterized by the undesired production of a protein. The organism can be contacted with an oligomeric compound which incorporates nucleosides prepared by the present methods. The oligomeric compound is synthesized to have a sequence that is capable of specifically hybridizing with a strand of target nucleic acid that codes for the undesirable protein.